We are developing a replication-competent Adenovirus (Ad)-recombinant priming/subunit boosting approach as a prophylactic vaccine for HIV/AIDS. Previous studies showing that the vaccine regimen can elicit humoral, cellular, and mucosal immunity together with significant protection in non-human primate models resulted in movement of this approach towards Phase I human trials. At the same time, we are conducting further pre-clinical studies to evaluate the utility of recombinants encoding additional viral genes, investigating novel subunit protein boosts, and exploring routes of immunization for elicitation of the best overall immune responses and protective efficacy. Recently studies in the SIV/Rhesus macaque model have shown that two sequential administrations of Ad5 host range mutant (Ad5hr)-SIV recombinants can boost SIV-specific immune responses, suggesting that modest antibody levels resulting from prior Ad-exposure might not preclude effective immunization. This study also showed the ability of Ad5hr-SIV recombinants encoding the env/rev and gag genes to effectively prime SIV-specific immunity, leading to partial protection during the acute phase and set point of infection following SIV challenge. A further study investigated complex Ad5hr-SIV recombinant priming regimens, including one encoding a non-myristoylated SIV nef, together with boosting by either gp120 or a peptide polymer representing the CD4 binding site of the envelope protein. Impressive results were obtained which showed that 39% of macaques immunized with recombinants encoding 3 or more SIV genes and boosted with gp120, or immunized with the Ad5hr-SIVenv/rev recombinant only but boosted with the peptide polymer, were highly protected. Following challenge with the highly pathogenic SIVmac251 isolate, viremia was either undetectable or it was cleared or controlled at the threshold of detection. Both cellular immunity and antibody responses appeared to contribute to this protective outcome. In a further comparative study, we showed that the better protection elicited by the second study was likely due to the route of vaccine administration, including an intranasal plus oral immunization followed by intratracheal administration of the Ad5hr-recombinants. This regimen elicited better secretory antibody responses, compared to the former study in which the intratracheal route was not used. Further studies investigating oral immunization with enteric-coated tablets to better deliver the Ad-recombinants to gut epithelium, thus targeting gut-associated lymphoid tissue, are planned. Overall, we have shown that the replicating Ad-recombinants elicit potent and persistent immune responses, including immunity to both dominant and subdominant cellular epitopes. A particularly novel finding was the observation of modulation of viral-specific cellular immune responses depending on the combination of Ad5hr-recombinants administered. The modulation effect did not persist through the period of protein boosting, however, suggesting that the replicating vector may be advantageous in overcoming any in vivo competition for elements of the immune system. In recent studies we have seen better cellular immune responses in non-human primates immunized with replicating recombinants in comparison to non-replicating Ad-recombinants administered at the same or higher dose. We believe the replication competent vector may be most advantageous for vaccine design, as it can achieve equivalent expression of inserted genes at lower doses, can more effectively target mucosal inductive sites, can better elicit cytokines and co-stimulatory molecules which serve as natural adjuvants, and has a greater potential for generating more persistent immunity.